This new project involves the study of a rapidly emerging group of immune receptors. Many families of inhibitory immune receptors have recently been identified in both mice and humans. Interestingly, within each of these inhibitory families of receptors, there are proteins that have lost the inhibitory domains. Instead these receptors have gained a positively charged amino acid within their transmembrane domain, suggesting that they may interact with signal transduction chains and transmit positive signals. In this project, we study the signal transduction and biochemistry of these putative positive regulators of immune cell function. Of nine described Ly49 molecules only 2, Ly49D and H, lack inhibitory domains and have charged transmembrane domains. Together with John Ortaldo's laboratory, we showed that Ly49D's transmembrane domain mediates an interaction with a newly described signaling chain, DAP12. Now this project involves the dissection of DAP-mediated signals. So far my laboratory has extensively characterized the signal transduction cascade of DAP12 in natural killer cells, including identification of several of the tyrosine phosphoproteins induced by its activation and demonstration that DAP12 signaling is coupled to the Syk tyrosine kinase, not Zap-70. Recently, a family of immunoglobulin superfamily inhibitory receptors, termed Paired Immunoglobulin-like Receptors (PIR), have been described in the B cells and macrophages of mice. This family also includes putative positive receptors that lack inhibitory domains and carry charged transmembrane domains, however, the biochemical mechanisms of these receptors are unknown. Because DAP12 is expressed in macrophages but Ly49s are not, several investigators have suggested that PIR may be the DAP12-associated receptors in these cells. Therefore, we have used a chimeric receptor approach to ask if PIR act through DAP12. These studies have determined that PIR physically interact with the gamma chain of the Fc epsilon receptor, but not DAP12, in murine macrophages. In addition, these data demonstrated for the first time that PIR can activate macrophages. However, they leave open the question of what receptors interact with DAP12 in macrophages. This and other questions regarding the biochemistry and signal transduction of DAP12 in the NK and macrophage lineages are the continuing focus of this project. These studies should help to elucidate the complex regulation of NK and macrophage function.